Ribonucleotide reductase is the enzyme responsible for the conversion of ribonucleotides to deoxyribonucleotides. As this is a crucial process in DNA synthesis, the enzyme is a favored target for anticancer and antiviral drug therapies. The long term goal of this research is to understand the catalytic mechanism of the enzyme for purposes of developing effective clinical treatments. This work focuses on determining the mechanism for cofactor assembly in the E. coli and mammalian enzymes. The catalytic cofactor of the R2 subunit of the enzyme is a diferric cluster in close proximity of a tyrosine radical. This radical is essential for initiating long range electron transfer to the second subunit (RI) where the substrate is reduced. Thus far, several intermediates have been identified in the assembly of the R2 cofactor from apoprotein, Fe2+ and 02. This work will continue the structural characterization of the intermediate X that can be trapped in the assembly process in E. coli enzyme using rapid freeze quench spectroscopies (EPR, Mossbauer, Raman and EXAFS). By altering the conditions during assembly (pH, temperature, mutants, or reactants), I hope to be able to accumulate a putative Fe3+-peroxo species. Finally, these studies on the well characterized E. coli enzyme will be extended to the lesser studied mammalian enzyme, as there are likely to be species dependent differences in the assembly process that may be clinically relevant.